The genotype-directed use of EGFR TKIs in EGFR-mutant lung cancer patients has fundamentally changed the face of the disease with improved response to therapy, preserved quality of life, and prolonged progression-free and overall survival. However, tumors that initially respond acquire resistance after about 1 year. In 50-60% of cases, resistance is mediated by the T790M resistance mutation in the gatekeeper location. However, we now appreciate that it may be overly simplistic to categorize cancers in a binary fashion such as T790M positive or negative. We have observed that within an individual patient there exist intra- tumoral and inter-tumoral heterogeneity with respect to T790M. Importantly, novel T790M-specific 3rd- generation EGFR inhibitors are currently entering the clinic and have shown significant activity in phase I trials for patients whose tumors harbor T790M. We have also learned that a subset of T790M positive cancers are intrinsically resistant to T790M inhibitors because they are no longer solely reliant on EGFR signaling for their viability. Thus, there is an increasing urgency to understand T790M heterogeneity, intrinsic resistance and their implications on patient response to these new therapies. In this proposal, we will tackle two aspects limiting the efficacy to 3rd generation EGFR TKIs; clonal heterogeneity and intrinsic lack of sensitivity. We will comprehensively explore clonal T790M heterogeneity in EGFR-mutant cancers with acquired resistance to the initial EGFR TKIs, and expand on our Preliminary Data suggesting that the amount of heterogeneity impacts response to 3rd-generation EGFR inhibitors. Utilizing cell lines derived directly from patient biopsies, we will also design and test therapeutic strategies to improve treatment outcomes in tumors that are inherently resistant to 3rd-generation TKIs. This study should lay the groundwork for newer diagnostic and treatment paradigms that assess, combat and overcome the complexities of targeted therapy resistance so patients can enjoy long-term remissions or even cures. Specific Aim 1: Define the intra- and inter-tumoral heterogeneity of T790M clones in cancers with acquired resistance to EGFR TKIs Specific Aim 2: Determine if T790M heterogeneity predicts responsiveness to 3rd generation EGFR inhibitors. Specific Aim 3: Identify therapeutic strategies for T790M cancers with intrinsic resistance to 3rd generation EGFR inhibitors.